Ion-detecting microspheres and methods of use thereof

ABSTRACT

This invention provides methods of using ion-detecting microspheres containing an ionphore and a chromoionphore in clinical laboratory instrumentation such as flow cytometry for sample analysis. In one embodiment, the microspheres are contacted with a flowing stream of a sample under conditions that allow the ion-selective ionophores to complex with the ions in the sample, and to cause deprotonation of the chromoionophore. The complexes are then exposed to an excitation wavelength light source suitable for exciting the deprotonated chromoionophore to emit a fluorescence signal pattern. Detection of the fluorescence signal pattern emitted by the deprotonated chromoionophore in microspheres containing the complexes allows for determination of the presence of the target ions in the sample. In one embodiment, lead ion-detecting microspheres are provided that can detect nanomolar levels of lead ions with response times on the order of minutes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a divisional of application Ser. No. 10/384,082 filed Mar. 7,2003, the entire contents of which are incorporated by reference. Thepresent invention claims priority to Provisional Application No.60/363,180, filed on Mar. 11, 2002, entitled “Rapid Trace Level SensingFluorescent Microspheres.”

GOVERNMENT INTERESTS

The invention was made in the course of work supported by grant No.GM59716 from the National Institutes of Health. The United Statesgovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to systems for detecting target ions ina sample, and more specifically, to ion-detecting microspheres andmethods of use thereof in clinical laboratory instrumentation such asflow cytometry for sample analysis.

2. Description of the Prior Art

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims.

The analysis of complex biological fluids, such as whole blood, serum,and urine, is of paramount importance in clinical chemistry.Electrolytes such as sodium ions and potassium ions are routinelyassessed using carrier-based ion-selective electrodes (ISEs) (1-2). Withmore than one billion ISE measurements being performed annually worldwide in clinical laboratories, this class of chemical sensors plays acrucial role in laboratory diagnostics. Trends in analytical chemistrycontinue to move toward the development of miniaturized systems, andthere is a great interest in streamlining all available assays into onecommon method. One approach towards achieving this goal is the use ofoptical readout methodologies.

Several approaches have been developed for ion analyses that usefluorescence transduction (3-17). This is primarily due to the highsignal-to-noise ratio afforded by this detection method, making it anattractive choice for creating sensors of reduced size. Mostion-selective optical sensors use the same carriers previously developedfor use in ISEs, and they obey bulk extraction principles consistentwith traditional optode theory (2).

Typically, an optode membrane is composed of a plasticized poly-(vinylchloride) (PVC) matrix, an ionophore that selectively binds the primaryion, ionic sites that facilitate mass transfer of ions from the aqueoussample to a hydrophobic sensing phase, and a hydrogen ion-selectivefluorescent chromoionophore (fluoroionophore), which is responsible forsignal transduction. Other approaches for ion determinations utilizedparticle-based technologies. Lubbers et al. have reported opticalnanoprobes for measuring the pH and pO₂ of physiological structures(10). More recently, Kopelman et al. reported both acryl amide andPVC-type nanometer-sized sensing spheres that have proven to be quiteuseful in interrogating intracellular environments (3, 4, 5, 19). Bakkerand group have prepared micrometer-sized particles by various methods,including heterogeneous polymerization (13), solvent casting (17), andvery recently, with the use of a sonic stream particle casting apparatus(16). Spatial and spectral characterization of the particles wasperformed by fluorescence microscopy/spectroscopy. However, the use ofthis technique has not been described for high throughput screeningapplications prior to the present invention.

In an attempt to increase the throughput of ion determinations, Kim etal. have described a 96-well plate-format absorbance-based optode thatrequires micro volume samples and that could be read using existingclinical laboratory instrumentation (20). An even more promisingtechnique, however, that offers rapid, high-throughput analyses withmultiplexing capabilities is flow cytometry. Microspheres have been usedfor flow cytometric applications for more than 25 years (21), and theyare commonly used for multiplexed analyses. It has been demonstratedthat as many as 64 different analytes can be screened simultaneouslyusing microsphere-based technologies (20-24). With large surface areaavailable for attaching numerous molecular recognition chemistries (6)and a core that can be impregnated with encoding dyes (25), microsphereshave played an important role in the development of suspension arraytechnologies (26). Numerous biologically relevant analytes have beendetected using microsphere-based cytometry (7, 8, 11, 12, 24, 27-29).Electrolytes, however, are a class of analytes that have never beenassessed with this technique.

Lower detection limits, smaller sample volumes, faster response timesand high selectivity are among the many requirements that must be met inthe trace level analysis of complex samples. Towards this end, optodefilms based on neutral ionophores have proven to be a highly promisingtechnology for the analysis of heavy metal ions. Over the past decade,an increasing number of cation-exchange based systems including thosefor Pb²⁺, Cu²⁺, Hg²⁺, Ag⁺, UO₂ ²⁺, have been reported (30-34).

For the analysis of lead ions, optode systems that incorporate highlyselective ionophores have been used. Those that may contain sulfurcoordinating functionalities, like a calixarene bearing a —SH pendinggroup or a di-thioamide derivative, have demonstrated increased successin polymer membranes of ISEs where they have been shown to exhibitdetection limits that extend even to picomolar levels with electrodesthat are carefully tailored (35, 36). In optode systems, selectiveionophores incorporated with a lipophilic chromoionophore and requiredanionic sites have been examined with view to their use in environmentalmonitoring (30). As for all conventional cation-exchange based systems,lead optodes follow predicted theory as given by the associatedequilibrium of transfer of the lead analyte species and hydrogen ionsinto the plasticized PVC optode phase. In particular, the leadcomplexing agent 3,6-dioxaoctanedithioamide derivative, that is, ETH5435, in conjunction with the absorption changing properties ofchromoionophore ETH 5418, was found to exhibit excellent selectivityagainst all relevant alkaline and alkaline-earth metal ions, thusallowing lead measurable concentrations to extend to the sub-nanomolarrange.

Antico et al. (37) have reported on optode films incorporating theionophore ETH 5493, which is a mono-thio oxodiamide derivative of ETH5435. While the use of such an ionophore offers less good selectivitywith respect to the alkaline-earth metal ions Ca²⁺ and Mg²⁺ than thedi-thioamide ligand, no irreversible sensing film poisoning uponexposure to Ag⁺ or Hg⁺ ions does occur.

These papers on ionophore-based optodes have shown that the selectivityand detection limits are sufficient to reach sub-nanomolar detectionlimits. However, a significant drawback is the high sensing volume ofthe optode film, which requires typically on the order of 10 μmoles ofions to be extracted from the sample in order to achieve the desiredoptical response. Consequently, massive volumes of sample (many liters)and very long response times (many hours) in a continuous flowing systemhave so far been required to accurately measure low levels of heavymetals in aqueous samples. A drastic miniaturization of the sensingelement should be able to alleviate this important problem.

SUMMARY OF THE INVENTION

Accordingly, this invention provides ion-detecting microspheres fordetecting target ions in a sample, and to methods of use thereof inclinical laboratory instrumentation such as flow cytometry for sampleanalysis. This invention is based on the discovery that ion-detectingmicrospheres can be used in conjunction with existing clinicallaboratory instrumentation such as flow cytometry as a diagnostic toolfor rapid, high-throughput analysis of a sample. The ion-detectingmicrospheres of this invention were shown to possess acceptablesensitivity, selectivity, and precision for the potential clinicaldetermination of ions. This invention further demonstrates thatmultiplexed measurements of two or more types of ions in a sample arepossible using this analytical method.

More specifically, one aspect of this invention provides a method ofdetecting target ions in a sample, comprising:

(a) providing polymeric ion-detecting microspheres, said microspherescomprising an ionophore selective for said target ions and achromoionophore;

(b) contacting said microspheres with a flowing stream of said sampleunder conditions that allow the ion-selective ionophores to bind andform complexes with the ions, if present in the sample, and to causedeprotonation of the chromoionophore;

(c) exposing the deprotonated chromoionophore, if formed, to anexcitation wavelength light source suitable for exciting thedeprotonated chromoionophore to emit a fluorescence signal pattern; and

(d) detecting the fluorescence signal pattern emitted by the complexes,if present, by a detection means for detecting the fluorescence signalpattern, wherein said fluorescence signal pattern is inverselyproportional to the amount of said target ions in said sample.

In one embodiment, the ion-detecting microspheres are immobilized on asubstrate prior to the contacting step.

This invention further provides rapidly responsive ion-detectingpolymeric microparticles for detecting metal ions in a sample. In oneembodiment, the ion-detecting microspheres comprise a lipophilicionophore selective for the metal ions, a chromoionophore selective forhydrogen ions, and a fluorescent dye, wherein said chromoionophorebecomes deprotonated when metal ions are present in said sample, andwherein the deprotonated form of the chromoionophore is absorbent at thefrequency of the fluorescence emission of the dye.

In particular, lead ion-detecting microspheres are provided that respondwith a high degree of sensitivity (i.e., can detect nanomolar levels oflead ions) and display extremely enhanced equilibrium response times(i.e., on the order of minutes). In addition, the lead ion-detectingmicrospheres only require sample volumes on the order of milliliters,and demonstrate high response stability for various lead ionconcentration changes.

This invention further provides a method of detecting nanomolar orsub-nanomolar levels of lead ions in a sample, comprising:

(a) providing polymeric microspheres comprising an ionophore selectivefor lead ions, a reference dye, and a chromoionophore selective forhydrogen ions, and a fluorescent dye, wherein the chromoionophore is achromoionophore that becomes deprotonated when lead ions are present inthe sample, and wherein said deprotonated form of said chromoionophoreis absorbent at the frequency of the fluorescence emission of said dye

(b) contacting said microspheres with a flowing stream of said sampleunder conditions that allow the ion-selective ionophore to bind and formcomplexes with the lead ions, if present in the sample, and to causedeprotonation of said chromoionophore;

(c) exposing the complexes, if formed, to an excitation wavelength lightsource suitable for exciting the fluorescent microspheres of thecomplexes to emit a fluorescence signal pattern; and

(d) detecting the fluorescence signal pattern emitted by the complexes,if present, by a detection means for detecting the fluorescence signalpattern, wherein said fluorescence signal pattern is inverselyproportional to the amount of lead ions in the sample.

DESCRIPTION OF THE FIGURES

The above-mentioned and other features of this invention and the mannerof obtaining them will become more apparent, and will be best understoodby reference to the following description, taken in conjunction with theaccompanying drawings. These drawings depict only a typical embodimentof the invention and do not therefore limit its scope. They serve to addspecificity and detail, in which:

FIG. 1 is a schematic showing the optical configuration of a flowcytometer according to one embodiment of this invention.

FIG. 2A shows the absorbance of the protonated (dashed line) anddeprotonated (solid line) forms of ETH 5294 in DMSO.

FIG. 2B shows the fluorescence emission characteristics of theprotonated (dashed line) and deprotonated (solid line) forms of ETH 5294(λ_(ex)=635 nm).

FIG. 3 is a single-parameter histogram depicting peak channelfluorescence intensity variations in type 1 sodium-selectivemicrospheres equilibrated in 10⁻¹ M (left peak) and 10⁻⁴ M (right peak)sodium samples containing 4 mM Tris, pH 7.4. Horizontal bars representthe full width at half-maximum, from which CVs were determined.

FIG. 4A shows the response and selectivity of type 2 sodium-selectivemicrospheres in sodium (open circles) and potassium (solid circles)sample solutions containing 4 mM Tris, pII 7.4.

FIG. 4B shows the response and selectivity of potassium-selectivemicrospheres in sodium (open circles) and potassium (solid circles)sample solutions containing 4 mM Tris, pH 7.4.

FIG. 5 shows the results of a parallel analysis of type 1sodium-selective microspheres (open circles) and potassium-selectivemicrospheres in the presence of sodium sample solutions containing 4 mMTris, pH 7.4.

FIG. 6 is the fluorescence spectra of a lead ion-selective optode filmafter equilibration with different lead concentrations in Mg(OAc)₂buffer at pH 4.6. The optode film contains lead ionophore Pb—IV, NaTFPB,DOC, PVC, and the ETH 5418 chromoionophore (basic form 717 nm)modulating the fluorescence emission of the lipophilic DiIC₁₈ dye (617nm).

FIG. 7 is the spatially resolved fluorescence spectrum of a singlesensing lead particle in its deprotonated form as observed with afluorescence spectrophotometric microscope. The spatial position ofhighest fluorescence intensity marks the middle of the microsphere.

FIG. 8 shows the response function of single sensing particles toPb(NO₃)₂ solutions in Mg(OAc)₂ buffer at pH 4.7 (closed circles) and pH5.7 (open circles). Dashed lines indicate the detection limit at each pHcalculated according to reference 42. Both curves were generated withlog K_(exch) of −2.4.

FIG. 9 shows the response time behavior for a single sensing particleimmobilized in a capillary flow cell at different lead concentrations atpH 4.7. Arrows indicate the moment of injection as lead solutions ofsmaller concentrations flow past the particles until equilibration ofthe signal is achieved.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides ion-detecting microspheres for detecting targetions in a sample, and to methods of use thereof in clinical laboratoryinstrumentation such as flow cytometry for sample analysis. Thisinvention is based on the discovery that ion-detecting microspheres canbe used in conjunction with existing clinical laboratory instrumentationsuch as flow cytometry as a diagnostic tool for rapid, high-throughputanalysis of a sample. More specifically, one aspect of this inventionprovides a method of detecting target ions in a sample, comprising:

(a) providing polymeric ion-detecting microspheres, said microspherescomprising an ionophore selective for said target ions and a fluorescentchromoionophore;

(b) contacting said microspheres with a flowing stream of said sampleunder conditions that allow the ion-selective ionophores to bind andform complexes with the ions, if present in the sample, and to causedeprotonation of the chromoionophore;

(c) exposing the deprotonated chromoionophore, if formed, to anexcitation wavelength light source suitable for exciting thedeprotonated chromoionophore of the complexes to emit a fluorescencesignal pattern; and

(d) detecting the fluorescence signal pattern emitted by the complexes,if present, by a detection means for detecting the fluorescence signalpattern, wherein said fluorescence signal pattern is inverselyproportional to the amount of said target ions in said sample.

The term “microsphere” or “microparticle” refers to a micrometer-sizedparticle which is comprised of a polymeric material which may include,but is not limited to, a thermoplastic (e.g., one or more ofpolystyrenes, polyvinyl chloride, polyacrylate, nylon, substitutedstyrenes, polyamides, polycarbonate, polymethylacrylic acids,polyaldehydes, and the like), latex, or acrylic. In one embodiment, themicrosphere is substantially spherical in shape.

In a preferred embodiment, the polymeric microspheres comprise amonodisperse population having an average particle size (as measured bydiameter) in the range of approximately 1 μm to about 20 μm.

Preferably, the microspheres are formed by a sonic stream particlecasting apparatus. Briefly, sonic stream particle casting is a method inwhich two liquids coexisting as two separate phases are brought incontact by flowing from separate storage reservoirs (38). One of theliquids, that is, the polymer solution mixture containing the chemicalsensing components (see below), is dissolved in an adequate organicsolvent that acts as the core stream. This solution and a second flowingaqueous solution, the so-called sheath liquid, are directed to a chamberwhere the constant oscillation frequency from a piezoelectric crystal,driven by a frequency generator (e.g, a BK Precision Model 4011,Placentia, Calif.) controls the formation of polymer droplets at a highrate. As the core polymer liquid flows under precisely definedconditions into an injection tube in the chamber and emerges as a liquidjet from a ceramic orifice tip (43 μm diameter), the polymer dropletsform within the second moving aqueous stream which flows around theinjection tube and emerging jet. Thus, the polymer droplet solventslowly partitions into the aqueous sheath stream leaving behind smalland highly uniform spherical particles that can be collected in arecipient solution for subsequent precipitation and separation, or canbe collected directly into small vials for immediate immobilization andanalysis. The casting process produces uniform polymeric microspheres ofaverage diameter-ranging between 3 and 30 μm.

Alternatively, the microparticles can be prepared by other methods knownto those skilled in the art, such as by a solvent casting method or byheterogeneous polymerization.

In one embodiment, uniform, monodisperse ion-detecting microspheres wereprepared from plasticized PVC using a high-throughput particle castingtechnique. Plasticized PVC was selected as the polymer matrix because itprovides a lipophilic environment conducive for retention of activesensing components, and it is known to be a suitable material forionophore-based sensing (1).

Alternatively, the microspheres comprise a copolymer of methacrylatemonomers with different pendant alkyl groups R₁ and R₂, wherein R₁ maybe any of C₁₋₃ alkyl group, and R₂ may be any of C₄₋₁₂ alkyl group, asdescribed in U.S. patent application Ser. No. 10/313,090, the content ofwhich is specifically incorporated in its entirety herein by reference.

The polymeric microspheres further comprise an ionophore having highselectivity for the target ion. The microspheres may be used inconnection with a wide variety of ionophores for detecting differenttarget ions. Examples of such ionophores include, but not are limitedto, ionophores selective for target ions such as hydrogen, Li⁺, Na⁺, K⁺,Ca²⁺, or Mg²⁺, metal ions such as Pb²⁺, Cu²⁺, Hg²⁺, Ag⁺, and oxides suchas UO₂ ²⁺. Examples of ionophores suitable for purposes of thisinvention include, but are not limited to, potassium ionophore III(BME-44), sodium ionophore X, and lead ionophore ETH 5493. Ionophores ofthe type described herein above are well known in the art arecommercially available or may be prepared using conventional synthetictechniques.

The polymeric microspheres also comprise a chromoionophore to allow forquantitation and/or detection of target ions in the sample, for exampleas described below with respect to flow cytometric methods. In oneembodiment, the chromoionophore is a neutral hydrogen-ion-selectivefluorescent chromoionophore (i.e., a fluoroionophore), which isresponsible for signal transduction. Deprotonation of thechromoionophore occurs when protons are exchanged by target ionsentering the polymeric matrix, and changes in chromoionophoreprotonation result in measurable changes in its optical behavior.

The ion-detecting sensors of the present invention may also includeother additives such as ion-exchangers to enhance the extraction of thetarget ion from the aqueous sample and the migration of the target ioninto the polymer matrix. While any ion exchangers that providelipophilic anionic sites on the polymer matrix may be used, preferably,carba-closo-dodecaborates, particularly halogenated carborane anions,are used as ion exchangers. Examples of halogenated dodecacarboranecation exchangers suitable for purposes of this invention include, butare not limited to, trimethylammonium-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12undecabromocarborane (TMAUBC) (U.S. patent application Ser. No.10/313,090), and salts (e.g., trimethylammonium salts) ofundecachlorinatedcarborane (UCC), hexabrominatedcarborane (HBC) andundecaiodinatedcarborane (UIC) anions.

It is a discovery of the present invention that ion-detectingmicrospheres possess acceptable sensitivity, selectivity, and precisionfor the determination of ions in a sample using existing clinicallaboratory instrumentation such as flow cytometry. For example, it wasdiscovered that ion-detecting microspheres such as those describedherein can be used in combination with flow cytometry as a usefulanalytical detection platform for rapid, high-throughput analysis ofsample ion concentrations.

FIG. 1 shows one example of a flow cytometry optical system usedaccording to this invention. According to FIG. 1, the microspheres arecontacted with a flowing stream of a sample under conditions that allowthe ion-selective ionophores to bind the ions, if present in the sample,and thus form a complex with the ions. As the ions form complexes withthe ionophore, the chromoionophore in the microsphere becomesdeprotonated. The microspheres are then exposed to an excitationwavelength light source suitable for exciting the deprotonatedchromoionophore to emit a fluorescence signal pattern. Detection of thefluorescence signal pattern emitted by the deprotonated chromoionophoreby a suitable detection means allows for determination of the presenceof the target ions in the sample as described below. The detection meanscan include, but is not limited to, a photodetector, a fluorimeter, afluorescence microscope, a filter, a charge couple device camera, or aphotomultiplier tube.

In one embodiment, the ion-detecting microspheres are immobilized on asubstrate prior to the contacting step. Examples of substrates include,but are not limited to, glass, silica, ceramic, nitrocellulose, nylon orother polymeric membrane materials. The substrate may be fabricated in aform of plates including multiple well microplates, sheets, films,slides, gels, membranes, beads, particles, foams, filaments, threads,and like structures. Methods of immobilizing the microspheres onto asubstrate may be performed as described herein or by methods known tothose skilled in the art.

According to one embodiment of this invention, a method is provideddetecting target ions in a sample, wherein the method includescontacting microspheres comprising an ionophore selective for the targetions with a flowing stream of a sample under conditions that allow theion-selective ionophores to bind and form complexes with the ions, ifpresent in the sample, and to cause deprotonation of thechromoionophore. The contacting conditions are those which allow theion-selective ionophores and target ions may become bound to each otheraccording to an ion exchange process consistent with classical optodetheory. In this process, target ions are extracted into the bulk of themicrosphere where they are complexed by the ion-selective ionophores.Influx of positive charge into the microsphere results in deprotonationof the fluoroionophore and a concerted expulsion of a proton from withinthe microsphere. The equilibrium describing this process is described byEquation 1:

I⁺(aq)+L(org)+CH⁺(org)+R⁻(org)=IL ⁺(org)+C(org)+R⁻(org)+H⁺  (1)

where I⁺ is the sample ion, L and IL⁺ are the uncomplexed and complexedforms of the ionophore, respectively, CH⁺ and C are the protonated anddeprotonated forms of the chromoionophore (e.g., a fluoroionophore),respectively, and R⁻ is the cation-exchanger (1). Parentheticalnotations (aq) and (org) denote the aqueous and organic phases,respectively. If the sample pH remains constant (i.e., via buffering),the concerted ion exchange allows for the quantitative determination ofthe target analyte by measuring changes in the optical activity of thechromoionophore (e.g., fluorescence intensity). Ionophore-mediatedsensing strategies are superior to sensing approaches that usesurface-attached indicators (6) because of the high selectivity impartedby the ion-detecting microspheres.

One example of a suitable chromoionophore for purposes of this inventionis the fluoroionophore ETH 5294 (30). The absorbance and fluorescenceemission characteristics of the ETH 5294 are shown in FIGS. 2A and 2B.In DMSO, the protonated form of ETH 5294 has absorption maxima at 280,324, and 635 nm, whereas the deprotonated form has maxima at 272, 514and 615 nm and a shoulder at 305 nm. It is apparent from FIG. 2A thatthe deprotonated form of the fluoroionophore does not strongly absorb at635 nm, which may explain the absence of a fluorescence signal for thisform of the indicator in FIG. 2B. The protonated form of thefluoroionophore, however, exhibits an emission maximum at 674 nm. Whenexcited at 635 nm, the emission behavior of ETH 5294 is substantiallydifferent from that observed when it is excited at 560 nm (17). A lossof ratiometric capabilities results, which is consistent with datareported for another self-referencing fluoroionophore (31). Thus, unlikemost fluorescence-based assays, which quantitate a directly proportionalrelationship between analyte concentration and fluorescence intensity,the approach used according to this invention uses an inverserelationship. That is, an observed decrease in fluorescence correlatesto an increase in ion concentration. This can be explained by Equation1, which shows that the deprotonated form of the fluoroionophorepredominates at higher target ion concentrations.

In one embodiment, microspheres selective for either sodium ions orpotassium ions were prepared according to the methods described inExample 1. Separate solution analyses of these microspheres resulted inthe generation of functional response curves using peak channelfluorescence intensities. FIG. 3 shows a single-parameter histogramdepicting peak channel fluorescence intensity variations in type 1sodium-selective microspheres (see Example 1) equilibrated in 10⁻¹ M(left peak) and 10⁻⁴ M (right peak) sodium samples containing 4 mM Tris,pH 7.4. This single-parameter histogram shows the number of counts as afunction of the log of the peak channel fluorescence. The horizontaldistance between the Gaussian curves, which represents sample sodiumconcentrations of 10⁻¹ M (left peak) and 10⁻¹ M (right peak), isindicative of a substantial change in the fluorescence behavior of thefluoroionophore. An increase in target ion concentration results in anincrease in the proportion of fluoroionophore in the deprotonated state,which does not emit (see FIG. 2).

Response curves depicting the fluorescence changes over a wide range ofsample concentrations for sodium ion-sensing microspheres and potassiumion-sensing microspheres are shown in FIGS. 4A and 4B, respectively.Type 2 sodium (i.e., particles containing a higher concentration ofsodium ion ionophore X as described in Example 1) were used to generatethe curves in FIG. 4A. The data points shown in FIGS. 4A and 4B are themean values of 2000 sensors. Separate sample solutions were used foreach respective ion. It was observed that higher concentrations ofpotassium were required to deprotonate the fluoroionophore, indicatingthe selective discrimination of potassium by the sodium ionophore. Aparallel shift in the location of the potassium response curve of about2.5 orders of magnitude is consistent with that previously reportedusing other detection platforms.

The above results demonstrate that ion-detecting microspheres can beused in conjunction with existing clinical laboratory instrumentationsuch as flow cytometry as a diagnostic tool for rapid, high-throughputanalysis of a sample.

When considering the utility of flow cytometry as a diagnostic tool forthe clinical determination of ions, one must consider whether thisapproach will work for analyzing complex biological fluids. A precursorto that step, however, is the parallel analysis of more than one type ofsensor. To test whether the ion-detecting particles of this inventionare useful for multiplex analysis, both sodium and potassium-selectiveparticles were suspended in samples containing various sodium ionconcentrations. Cytometric analysis of the mixed particle suspensionresulted in a single region of particle density on a forward-scatterversus side-scatter plot, which implies homogeneity of particle size(data not shown). The region of particle density was gated and analyzedusing FL2 which separated the particle subsets into two distinct regionson the basis of their long-wavelength fluorescence behavior. Because thetype 1 sodium particles were also doped with a long-wavelength activedye, particle subsets could easily be segregated. Each particle subsetwas then gated and analyzed using FL1, which is the fluorescence channelcorresponding to the fluorescence signal generated by ETH 5294. Theresulting response curves are shown in FIG. 5.

As shown in FIG. 5, a functional response curve was obtained for sodium(open circles), whereas the curve for potassium (closed circles) isindicative of the selectivity behavior of the potassium ionophore BME-44over sodium. The selectivity behavior of sodium ionophore X agrees withthe results shown in FIG. 4A, in which the microspheres were measured ina serial manner (i.e., each particle subset was measured separately).Preliminary lifetime studies of these microsphere-based sensors haveshown that type 2 sodium particles have remained fully functional forperiods as long as five weeks. This first step toward the clinicaldetermination of ions lays the foundation for the potentialapplicability of this technology for the multiplexed analysis of complexbiological fluids.

According to another aspect, this invention provides metal ion-selectivepolymeric microspheres for detecting metal ions in a sample, saidmicrospheres comprising a lipophilic ionophore selective for said metalions, a chromoionophore selective for hydrogen ions, and a fluorescentdye, wherein said chromoionophore is a chromoionophore that becomesdeprotonated when said metal ions in said sample are present, andwherein said deprotonated form of said chromoionophore is absorbent atthe frequency of the fluorescence emission of said dye.

Examples of metal ion-selective polymeric microspheres of this inventioninclude microspheres selective for metals such as Pb²⁺, Cu²⁺, Hg²⁺, Ag⁺,as well as oxides such as UO₂ ²⁺. The metal ion-selective polymericmicrospheres can include any cation exchange-based systems includingthose for Pb²⁺, Cu²⁺, Hg²⁺, Ag⁺, UO₂ ²⁺, such as those reported in theliterature (30-34). A preferred lead ionophore isN,N,N′,N′-tetradodecyl-3,6-dioxaoctane-1-thio-8-oxodiamide (ETH 5493).

The metal ion-selective microspheres of the present invention furtherinclude a chromoionophore. Examples of chromoionophore include, but arenot limited to, a pH indicating chromoionophore, a chromoionophore, afluoroionophore, a pH indicator, or a pH indicating fluoroionophore.

In one embodiment, metal ion-selective microspheres of this inventioncomprise a lipophilic ionophore selective for lead ions, and achromoionophore selective for hydrogen ions, incorporated in a polymermatrix. For such a composition, the lead ion activity (a_(Pb)) of themicrospheres is dependent on the pH of the sample. The lead ion activityis also dependent on the molar concentration of active components,namely, the total concentration of ionophore (L_(T)), the totalconcentration of chromoionophore (C_(T)), and the total amount oflipophilic anionic sites (R_(T−)). The value a, which corresponds to therelative fraction of unprotonated chromoionophore, is also introduced inthe following expression to describe the metal ion activity (a₁) in thesample according to Equation 2:

a ₁ =K _(exch) ⁻¹·(αa _(H)/1−α)^(z)·[(R _(T)−(1−α)·C ^(T))/z(L _(T)−n/z{R _(T)−(1−α)·C _(T)})^(n)]  (2)

where z is the charge of the analyte I^(+z) and n is the ion-ionophorecomplex stoichiometry. Most typically, the degree of protonation of thechromoionophore (1−α) is used to represent the response function ofoptode systems in absorbance mode.

Given the need to spatially and spectrally characterize microspheres viafluorescence microscopy, a judicious choice of the chromoionophore isrequired. The chromoionophore must display appreciable fluorescentquantum efficiency upon pH changes and it should also exhibit a pKavalue that permits an effective shift of the dynamic range and detectionlimit of lead ions to best-suited levels. Furthermore, it must ideallylend itself to ratiometric measurement in order to minimize any possiblephotobleaching effects and variations in positioning, size and lightintensity.

A first chromoionophore explored was the azo-derivative ETH 5315. ThepK_(a) value of ETH 5315 in optode membranes is 5.5 (39), and thereforeappeared suitable for detecting lead ions in an acidic media. However,ETH 5315 proved to be unsuccessful, as demonstrated by its very poorfluorescence emission when incorporated in optode films.

Alternatively, the more basic chromoionophore ETH 5418 (pK_(a) 8.8),which has been used in absorption-based measurements for lead and otherheavy metal ions, was investigated as a chromoionophore for the lead ionsensing microparticles of this invention. However, the basic form of ETH5418 is not light emitting. Therefore, the use of ETH 5418 forfluorescent-based measurements required the development of an “innerfilter” approach as follows.

It is known that the fluorescence spectral characteristics of DiIC₁₈ (alipophilic dye) can be modulated by the absorption changing propertiesof more basic chromoionophores (14). Accordingly, DiIC₁₈ was added tothe formulation containing the basic chromoionophore ETH 5418 for theproduction of the lead ion selective microparticles of this invention.It was discovered that the basic form of the chromoionophore ETH 5418absorbs strongly in the region where the reference dye DiIC₁₈ isfluorescent but where the acidic form of the chromoionophore exhibits nospectral characteristic.

FIG. 6 shows the spectral fluorescence response of an optode filmprepared with microspheres containing the chromoionophore ETH 5418, themodulator dye DiIC₁₈, the lead ionophore Pb—IV and the ion exchangerTFPB as a function of various lead ion concentrations in Mg(OAc)₂buffered sample at pH 4.7. As seen from FIG. 6, the ETH 5418-DiIC₁₈ pairlends itself nicely to spectral resolution of both emission peaks. Asdeprotonation of ETH 5418 (717 nm) occurs in response to lead activityin the sample the absorbance of its basic form increases sharply. As aconsequence of the overlap with the emission spectrum of DiIC₁₈, theemission of the DiIC₁₈ decreases appropriately.

Since the basic form of the chromoionophore is absorbent at thefrequency of the fluorescence emission of the DiIC₁₈, the fluorescenceintensity of the dye decreases as deprotonation of 5418 increases inresponse to an increase of lead ion concentration. Thus, the degree ofprotonation of the fluorescent chromoionophore is described as afunction of the observed fluorescence ratios as shown in Equation 3:

1−α=[IndH⁺]/Ind_(T)=1−[1+((R_(max)−R)/R—R_(m))]⁻¹   (3)

where R, and R_(min), and R_(max) are the fluorescence intensity ratiosfor a given equilibrium and at maximum and minimum protonation of thechromoionophore (10).

The lead ion-detecting microspheres whose response is depicted in FIG. 6were prepared with the casting apparatus as described in Example 3, inwhich a batch of PVC-DOS plasticized particles of equal composition tothe optode film incorporated the lead ionophore Pb—IV and the ETH 5418chromoionophore. However, although single particles were obtained whoseshape, size and acid-base response were as expected, exposure to dilutebuffer solutions resulted in complete or partial deprotonation of thechromoionophore, thus yielding irreproducible results. The very highselectivity coefficients of Pb—IV ionophore reported for polymericmembranes of ISEs (36) supported the fact that interference by bufferions was unlikely. Instead, the loss of Pb—IV ionophore during thecasting process was the most likely explanation. Efforts to greatlyincrease the concentration of Pb—IV ionophore were hindered by thelimited solubility of Pb—IV ionophore in DOS plasticizer Indeed, filmscontaining about 20 mmol/kg of ionophore where observed to crystallizein the membrane over time.

Recent reports on the use of Pb—IV ionophore in membranes of ISEsplasticized with o-NPOE demonstrating lead ion response in the nanomolarconcentration level (40) prompted the formulation of batches with thisplasticizer. However, in this case such efforts were met with theinability to deprotonate the chromoionophore, which may be indicative ofnear-complete leaching of the more-polar plasticizer (the particleproduced were significantly smaller than with DOS under the sameconditions).

As a result of these initial studies, the lead ion-selective ionophoreETH 5493 (37) was investigated as an ionophore for the leadion-detecting microspheres of this invention. Since the solubility ofthis ionophore in various plasticizers is not a problem at highconcentrations, particle batches as described in Example 3 wereprepared. Optical ion exchange constants (Equation 1) and selectivitycoefficients for particles prepared by incorporating this ionophore weredetermined for some relevant ions. The results are shown in Table 1(complex stoichiometries are from the reference (37)). As shown, theparticles display excellent selectivity characteristics, which is inline with earlier described data (37).

TABLE 1 Selectivity of lead-selective microsphere sensing particles.^(a)Ion z n^(b) log^(K) _(exch) log^(K) _(IJ) ^(opt) Pb²⁺ 2 2 −2.3 0 Na⁺ 1 1−3.6 −5.2 Ca²⁺ 2 2 −7.0 −10.9 Cd²⁺ 2 2 −1.2 1.0 K⁺ 1 1 −3.5 −5.1^(a)Procedure according to the references. (30, 41) ^(b)n is the assumedcomplex stoichiometry (37) and ^(K) _(exch) the experimentallydetermined ion-exchange constant for the listed ion (see eq 1).

FIG. 7 depicts a typical spatially resolved fluorescence spectrumobtained from a single lead ion-detecting microsphere with thechromoionophore ETH 5418 in its deprotonated form, lead ionophore ETH5493 (87.2 mmol/kg), chromoionophore ETH 5418 (10.2 mmol/kg),ion-exchanger NaTFPB (23.8 mmol/kg), and reference dye DiIC18 (6.9mmol/kg ) in PVC plasticized with DOS in a 1:2 ratio by weight,. Theparticles were found to be uniform in size and spherical, and theirsurface appeared smooth under the microscope.

FIG. 8 shows typical responses of the lead ion-detecting microspheres(same formulation as described with respect to FIG. 7) obtained for leadion concentrations ranging from 5×10⁻¹⁰ to 5×10 M⁻³ at pH 4.7 and pH5.7. As shown, the particles responded with a high degree of sensitivityand in full agreement of the expected theoretical values calculated onthe basis of Equation 3. Furthermore, the results emphasize theexcellent particle-to-particle reproducibility that is obtained via theparticle casting method, as the relative standard deviation of themeasurements is within a few percent (see error bars in FIG. 8).

FIG. 9 shows typical response times of a single lead ion-detectingmicrosphere (same formulation as described with respect to FIG. 7)immobilized in a glass capillary cell and assessed in the flowing streamof test samples containing lead ions. The arrows in FIG. 9 represent theapparent moment of injection in which the sensing particle firsttransitions from lower to higher concentrations of lead. As seen in FIG.9, the equilibrium response time for a 13 μm particle at lead ionconcentrations of 5×10⁻³ M and higher is reached essentially in 2.5minutes. For concentrations at detection limit levels, that is, about5×10⁻⁹ M, the time needed for complete response is about 15 minutes.FIG. 9 also illustrates the high response stability observed for thevarious concentration changes, thus indicating minimum or negligibledrift following multiple exposures to the light source.

Equilibrium response values on the order of minutes as demonstrated bythe lead ion-detecting microspheres of this invention represent aconsiderable enhancement over the response time behavior of optodefilms, which have been found to exhibit extremely long response times inhighly diluted samples. Indeed, optode membranes proposed earlier forthe analysis of Pb²⁺ at the nanomolar level necessitated hours ofequilibration for stable signals to be obtained (30). More recently,optode systems developed for other environmentally relevant ions e.g.,Ag²⁺ have also been shown to require response times in excess of fewhours at detection limit levels or tens of minutes for higherconcentrations. As the rate-limiting step in optode films in contactwith dilute solutions is thought to depend on the convective masstransport to the membrane, the saturation of the bulk equilibrium isexpected to be massively shortened for micron-size particles of the typedescribed herein. Such behavior clearly arises from the drasticallyreduced amount of active components found within the particle. Indeed, a10 μm particle requires only on the order of 2×10⁻¹¹ mol lead ions toreach an accurate optical response (at α=0.5), which is a full 4 ordersof magnitude smaller than with traditional thin films. This exemplifiesone of the benefits of miniaturization of this powerful chemistry.

The ion-detecting microspheres of the present invention may be used fordetecting ions of all types of body fluid samples. Examples of thesamples include, but are not limited to, whole blood, spinal fluid,blood serum, urine, saliva, semen, tears, etc. The fluid sample can beassayed neat or after dilution or treatment with a buffer.

The ion-detecting microspheres of the present invention may also be usedfor detecting ions of all types in environmental samples such as water.

Additional features and advantages of this invention shall be set forthin part in the description that follows, and in part will becomeapparent to those skilled in the art upon examination of the followingspecification or may be learned by the practice of the invention. Theadvantages and novel features of this invention may be realized andattained by means of the instrumentalities, combinations, and methodsparticularly pointed out in the appended claims.

EXAMPLES

The following reagents were used in the examples described below.Poly(vinyl chloride), (PVC), bis(2-ethylhexyl) sebacate (DOS),tert-butylcalix[4|arene tetraethyl ester (sodium ionophore X),2-dodecyl-2-methyl-1,3-propanediylbis[N-[5′-nitro-(benzo-15-crown-5)-4-yl]carbamate (potassium ionophoreIII, BME-44),9-(diethylamino)-5-octadecanoylimino-5H-benzo[a]phenoxazine(chromoionophore 1, ETH 5294), and sodiumtetrakis-[3.5-bis(trifluoromethyl)phenyl]borate (NaTFPB),4-tert-butylcalix[4]arene-tetrakis(N,N-dimethylthioacetamide) (leadionophore IV),11-[(-butylpentyl)oxy]-11-oxoundecyl4-[9-(dimethylamino)5H-benzi[a]phenoxazin-5yl-idene]aminobenzoate(ETH 5418, chromoionophore VH),4-[[9-dimethylamino]-5H-benzo[a]phenoxazin-5ylidene]amino]benzeneaceticacid 11-[(1-butyl-pentyl)oxy]-11-oxoundecylester (ETH 2439chromoionophore II), and tetrahydrofuran (THF) were Selectophore qualityfrom Fluka (Milwaukee, Wis.).

The ring-locked cyanine (RLC) reference dye2-[2-[2-chloro-3-[(1,3-dihydro-3,3-dimethyl-1-decylbenzoindol-2-ylidene)ethylidine]-1-cyclohexn-1-yl[ethenyl]-3,3-dimethyl-1-decylbenzoindoliumniodide was obtained from Beckman Coulter, Inc. (Brea, Calif.).Dichloromethane (DCM) (EM Sciences), xylenes (EM Sciences),cyclohexanone (J T Baker), and diethyl sulfoxide (DMSO) (Aldrich) wereACS grade and were obtained from the indicated suppliers. Chloride saltsof sodium and potassium were reagent grade and were obtained fromMallinckrodt and Sigma, respectively. Tris(hydroxymethyl)amino methane(Tris) was reagent grade from Sigma. A reverse-osmosis water filtrationsystem (US Filler, Philadelphia, Pa.) was used to distill and deionizethe water (18 MΩ) from which sample solutions were prepared.

The nitrate salts of lead, cadmium, copper, silver, mercury and thechloride salts of sodium, potassium and magnesium were all puriss. p. a.from Fluka. The internal reference dye1,1″-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiIC₁₈) was from Molecular Probes (Eugene, Oreg.), cyclohexanone(99.8%) was from Aldrich, and dichloromethane and xylenes (ACS grade)were from Fisher. The 4-(octadecylamino)azobenzene (chromoionophore ETH5315) was synthesized according to a published procedure (14). TheN,N,N′,N′-tetradodecyl-3,6-dioxaoctane-1-thio-8-oxodiamide (ETH 5493)ionophore was a gift from the laboratory of Prof. E. Pretsch (ETH,Zurich), which had been synthesized as described (37).

Example 1 Preparation of Ion-Detecting Microspheres

A polymer cocktail containing PVC, DOS, ionophore, ETH 5294 and NaTFPBwas dissolved in 5 mL of cyclohexanone and diluted with 100 mL ofdichloromethane. The addition of 1 mL of xylenes to the solution aidedin the aesthetic appearance of the cast particles. Potassium sensingmicrospheres contained PVC (33 wt %), DOS (66 wt %), ETH 5294 (0.2mmol/kg), NaTFPB (0.3 mmol/kg), and the potassium ionophore BME-44 (21.1mmol/kg). Sodium sensing microspheres contained PVC (33 wt %), DOS (66wt %), ETH 5294 (0.2 mmol/kg), and either sodium ion-ionophore X (10.0mmol/kg) and NaTFPB (0.3 mmol/kg) (type 1) or sodium ion-ionophore (89.3mmol/kg) and NaTFPB (27.1 mmol/kg) (type 2). For segregation of particlesubsets in the parallel analyses, type 1 sodium sensing particles alsocontained RLC reference dye (3.9×10⁻³ mmol/kg). Particles were preparedusing a particle casting apparatus as described by Tsagkatakis et al.(16).

The following settings and specifications were used in this example: tipdiameter: 38.1 μm; frequency, 21.S kHz; polymer flow rate, 0.5 mL/min:water flow rate, 52 mL/min: surfactant flow rate, 1 drop/10 s; curingduration, 4 days. The surfactant was added using a model 352 syringepump (Sage Instruments, Boston, Mass.).

Example 2

Instrumentation and Measurement

The absorption behavior of ETH 5294 in both its protonated anddeprotonated forms was determined using a DU 70 spectrophotometer(Beckman Coulter, Inc., Fullerton, Calif.). For fluorescencecharacterization, a 1×10⁻⁵ M solution of ETH 5294 in DMSO was mixed witha 1% (v/v) aqueous solution of either HCl or NaOH and excited at 635 nmusing a Fluorolog 3 fluorometer (Instruments SA, Edison, N.J.) todetermine the emission behavior of the protonated and deprotonated formsat this wavelength. Optical characterization of RLC in methanol foundλ_(ex)=820 nm and λ_(em)=860 nm.

A Beckman Coulter EPICS XL flow cytometer modified for both 635-nm and785-nm excitation was used to interrogate the sensing microspheres. A650-nm long-pass emission filter and a 660 (±15) nm band-pass filterwere used to collect fluorescence emitted between 650 and 675 nm, and an800-nm long-pass emission filter was used to collect fluorescenceemitted above 800 nm. A schematic representation illustrating theoptical setup of the cytometer that was used appears in FIG. 1. Thesensing microspheres were equilibrated for 30 seconds in sodium orpotassium sample solutions containing 4 mM Tris buffer, pH 7.4.

Example 3 Preparation of Lead Ion-Detecting Microspheres

The schematic set-up and the general protocol for the preparation ofsensing particles have been reported recently (17) and it applies herewith minor modifications. Unless otherwise indicated a cocktail mixturewas prepared by weighing out 58.5 mg PVC, 116 mg DOS, 1.44 mg (10.2mmol/kg) chromoionophore, 1.27 mg (6.9 mmol/kg) reference dye DiIC18,4.05 mg (23.8 mmol/kg) ion exchanger NaTFPB, 14.8 mg (87.2 mmol/kg)ionophore and dissolving it in 5 mL of cyclohexanone. The mixture wasshaken in a vortex mixer for approximately one hour and then addeddropwise to 100 mL dichloromethane under gentle stirring. After adding 1mL xylenes the solution was filtered through a 0.45 μm Gelman filter and50 mL transferred to a gas-tight Hamilton syringe. The syringecontaining the polymer core solution was mounted on a syringe pump(Stoelting, Wood Date, Ill.) and set to flow at a rate of 0.263 mLmin⁻¹. Deionized water used as the sheath liquid stream flowing at arate of 43 mL/min was controlled via a pressure regulator. The frequencygenerator was operated at a setting of 12.3-12.7 kHz.

Example 4 Instrumentation for Analyzing Lead Ion-Detecting Microspheres

A Pariss Imaging Spectrometer (Light Form, Belle Mead, N.J.) combinedwith a Nikon Eclipse E400 microscope with an epifluorescence attachment(Southern Micro Instruments, Marietta, Ga.) was used to opticallycharacterize optode films and particles. The system was equipped withtwo CCD cameras EDC 1000 L (Electrim Corp., Princeton, N.J.) and a Nikonsuper high-pressure mercury arc lamp (Southern Micro Instruments). Afilter cube with a 510-560 nm excitation filter, a 565 nm dichroicmirror, and a 590 nm long pass emission filter was used. The system,equipped with a motorized stage (Prior Optiscan ES9, Fulbourn, Cambs,UK.) was operated via Pariss data acquisition software, to recordindividual fluorescence spectra of particles and films under the fieldof view. The schematic representation of this optical arrangement hasbeen reported previously (17).

Example 5 Measurements of Lead Ion-Detecting Microspheres

Particles were collected in 20 mL small glass vials directly from theemerging jet of the casting apparatus. A 100 μL aliquot of liquidcontaining resuspended particles was deposited on a 22 mm wideFisherbrand® microscope cover glass immediately after collection andallowed to evaporate in the dark under a hood draft. After evaporation,the glass substrate containing immobilized particles was mounted into aflow cell, fitted on the motorized stage of the microscope and connectedto a small peristaltic pump (Dakota Instruments). Fluorescence spectrawere taken under static conditions and after equilibrating the flow cellwith given test concentrations. For response time measurements, aborosilicate glass microcapillary cell of 1.0 mm i.d. and 0.15 mm wallthickness was used in order to reduce the dead volume. A 50 μL aliquotof collected particles was pipetted inside the capillary cell and theliquid was left to evaporate under gentle vacuum. The capillary waspositioned over a glass fixture, attached at each end with polyethylenetubing and connected to a peristaltic pump operated at a rate of 0.1mL/min. Measurements were taken every 30-60 seconds for 5 minutes andevery minute or longer thereafter. In all cases, the image of a particlewas obtained using a 40×-microscope objective to capture a specificslice under the field of view, typically using transmission mode toavoid photobleaching. The spectral image of this slice was then taken inthe fluorescence mode. For all measurements neutral density filters 4and 8 were used to decrease the light intensity from—the source.Exposure time for spectral acquisition of particles was 500 ms and forfilms 300 ms.

Standard 5×10⁻³ M stock solutions were prepared by dissolving the metalsalt in a 1 mM magnesium acetate buffer of desired pH. Test solutionswere then prepared by stepwise gravimetric dilution with the samebuffer. For concentrations below 10⁻⁷ M, aliquots of the 10⁻⁶ M solutionwere diluted with increasing volumes of buffer to give the finalconcentration. Calibrating solutions were all prepared in polyethylenebeakers that had been pretreated with 0.01 M HNO₃.

Example 6 Preparation of Optode Films Comprising Lead Ion-DetectingMicrospheres

For optode film preparation, ion-exchanger salt additive (7.4 mmolkg⁻¹), lead ionophore (13 mmol kg), chromoionophore ETH 5418 or ETH 2439(6.5 mmol kg⁻¹), internal reference dye DiIC₁₈ (5.4 mmol kg⁻¹),plasticizer and PVC (2:1 by weight) were weighed out and dissolved in1.5 mL THF. After complete dissolution, the sensing cocktail was spincoated onto quartz glass plates and any remaining solvent was left toevaporate in a hood draft for at least 30 minutes prior to measurements.

Results and Discussion

Uniform, monodisperse ion-detecting microspheres were prepared fromplasticized PVC using a high-throughput particle casting technique (16).In one embodiment, the particles incorporated with an ionophore havinghigh selectivity for the target ion, and a neutral hydrogen-ionfluorophore.

Cytometric measurement of the fluorescence intensities from individualparticles produces a coefficient of variation (CV) in the estimate ofthe mean fluorescence equal to the CV of the particle population. The CVin the estimate of the mean can be improved by averaging themeasurements from multiple particles. Repeat subsampling of a read of10, 000 particles gave precision improvement consistent with Polsonstatistics. For mean peak fluorescence channel values of 109.3, the CVin the estimate of the mean improved from 12.5% to 1.2% to 0.4% when thesample size was increased from 1 to 300 to 1000, respectively.Conversely, potassium-selective particles containing the ionophoreBME-44 demonstrate function and selective discrimination of sodium ionsas shown in FIG. 4B. The selectivity observed, which is indicated by the2.5 orders of magnitude parallel shift of the sodium response curve, isconsistent with the particle-based detection method described byTsagkatakis et al. (17).

When dealing with biological samples, a primary concern is the biasingof results due to sample autofluorescence. This obstacle may becircumvented by judiciously selecting a fluoroionophore that hasappropriate spectral characteristics and by using long-wavelengthexcitation. Under these circumstances, the emission behavior of thesensing ionophore is usually beyond the wavelength range over whichautofluorescence typically occurs (3). Furthermore, encoding dyes suchas RLC and optical filters may also be used to eliminate undesiredfluorescence wavelengths.

The precision of microsphere-based flow cytometry is ultimatelycontingent upon the monodispersity of the particles. The particlefabrication method used herein has been reported to produce uniformparticles with a diameter variation of 1.5% (32). Furthermore, CVsrepresenting the particle-to-particle reproducibility of fluorescenceflow cytometric measurements have been reported to be as low as 3.6%(32), although this signal variation may appear to be a potentiallimitation of this technique, ratio metric capabilities of the readoutwill greatly improve reproducibility. Moreover, the ease of measurementof the numbers of particles makes excellent precision possible, evenwith poor particle uniformity.

Implementation of microsphere-based technologies for ion analyses makesit possible to use existing instrumentation commonly found in theclinical laboratory to determine this class of analytes. A suitabletechnique that is commonly used for microsphere-based assays is flowcytometry. Flow cytometry offers a means for high-throughput screening,which is a capability not found with microscopic techniques. This isprimarily due to differences in the detectors commonly employed witheach technique. In fluorescence microscopy, charge-coupled devices thatoffer excellent spatial and temporal resolution are typically used,however these devices suffer from longer data acquisition times on theorder of the millisecond time scale. Flow cytometry, on the other hand,uses photo-multiplier tubes, which allows for measurements to becollected on the microsecond time scale (29). This inherent advantage inconjunction with multiplexed measurement capabilities makes flowcytometry an attractive analytical method for clinical applications.

Conclusion

This invention demonstrates the applicability of flow cytometry as ameans for developing multiplexed, rapid, high-throughput analyses forclinically relevant ions. In one embodiment, ionophore-mediatedmicrosphere-based sensors selective for sodium and potassium wereprepared using a high-throughput particle casting apparatus thatutilizes a sonic droplet formation process. Flow cytometry wasdemonstrated to be a useful analytical detection platform for theanalysis of sample ion concentrations The ion sensing microspheres wereanalyzed serially and in parallel via long-wavelength flow cytometry andwere shown to possess acceptable sensitivity, selectivity, and precisionfor the potential clinical determination of these ions.

In another example, fluorescent plasticized PVC microspheresincorporating a selective ionophore for lead metal ions were preparedvia a casting apparatus that allows for the mass production of highlyreproducible spherical particles. Immobilized particles assayed in aflowing stream of analyte were imaged via fluorescence spectroscopy andshown to follow predicted theory, allowing lead ion determinations atthe low nanomolar level. Responses were characterized by high stabilityand reproducibility with a detection limit comparable to those found foroptode films. However, in contrast to optode thin films that requiretypical equilibration times of hours following exposure to nanomolarlevel concentrations, the particles prepared here were shown to respondrapidly in just a few minutes and required drastically reduced samplevolumes on the order of one milliliter. Miniaturized ionophore-basedsensing microspheres are therefore a very promising platform for theassessment of trace level concentrations in a variety of samples.

The present invention may be embodied in other specific forms withoutdeparting from its essential characteristics. The described embodimentis to be considered in all respects only as illustrative and not asrestrictive. The scope of the invention is, therefore, indicated by theappended claims rather than by the foregoing description. All changes,which come within the meaning and range of the equivalence of theclaims, are to be embraced within their scope.

The words “comprise,” “comprising,” “include,” “including,” and“includes” when used in this specification and in the following claimsare intended to specify the presence of stated features, integers,components, or steps, but they do not preclude the presence or additionof one or more other features, integers, components, steps, or groupsthereof.

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1-23. (canceled)
 24. Ion-detecting polymeric microspheres for detectingmetal ions in a sample, said microspheres comprising a lipophilicionophore selective for said metal ions, a chromoionophore selective forhydrogen ions, and a fluorescent dye, wherein said chromoionophore is achromoionophore that becomes deprotonated when said metal ions arepresent in said sample, and wherein said deprotonated form of saidchromoionophore is absorbent at the frequency of the fluorescenceemission of said dye.
 25. The polymeric microspheres of claim 24,wherein said ions are lead ions and said ionophore isN,N,N′,N′-tetradodecyl-3,6-dioxaoctane-1-thio-8-oxodiamide (ETH 5493).26. The ion-detecting sensor of claim 25, wherein said chromoionophoreis11-[(butylpentyl)oxy]-11-oxoundecyl-4-[9-(dimethylamino)5H-benzi[a]phenoxazin-5-yl-indene]aminobenzoate(ETH 5418) and said dye is1,1″-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate(DiIC₁₈).
 27. The polymeric microspheres of claim 24, wherein saidpolymer further comprises an ion-exchanger.
 28. The polymericmicrospheres of claim 24, wherein said particles are obtained by anautomated casting process. 29-31. (canceled)